3o6 DISINFECTANTS AND ANTISEPTICS 



If the results were as follows, 60 would be the figure employed. 



Only resistant cultures are admissible in the test, for the use of weak strains leads 

 to erroneous conclusions.' As old-stock strains rarely meet these requirements, it is 

 necessary to secure a freshly isolated culture. After isolation, if directions already 

 given for B. typhosus are followed, the resistance to phenol should be maintained for 

 at least a year. 



B. DIPHTHERIAE 



A preliminary comparison of freshly isolated strains of this organism with a stock 

 culture of Park No. 8 showed that all strains used possessed about equal resistance 

 to phenol. Since Park No. 8 proved so satisfactory as a test organism in a few prac- 

 tical tests and in repeated tests with phenol, it was selected as our stock organism for 

 this purpose, and so long as it continues to retain resistance comparable to that ex- 

 hibited by new isolations, it will be satisfactory as a test organism. 



The method used at the present time is: 



Three or four slants of Loeffler's blood serum, made from Difco dehydrated Loeffler's 

 blood serum, are streaked heavily with B. diphtheriae and incubated at 37° C. for 24 hours; 

 this is repeated for three consecutive days. The growth is then taken up in sterile saline and 

 made up to a density corresponding to nephelometer 2. Five-tenths cc. of this suspension is 

 then added to 5 cc. of the diluted disinfectant at 20° C. following the technique given for B. 

 typhosus and Staph, aureus. After 5, 10, and 15 minutes, one loopful of the mixture of culture 

 and diluted disinfectant is transferred to the Loeffler's slant, first dipping it into the liquid 

 at the base of the medium and then streaking over the slant. Another transfer is then made 

 from the liquor of the first tube and streaked on to a second tube of LoeflSer's medium. In 

 this way further dilution of the disinfectant carried over is assured. Subculture tubes are 

 then incubated at 37° C. for 48 hours, when they are observed for typical colonies of B. 

 diphtheriae. The slants showing growth from the most concentrated dilutions are con- 

 firmed by stained microscopic smears. Two phenol controls, i-ioo and 1-120, are included in 

 each test. B. diphtheriae under the conditions outlined above should not be killed by i-ioo 

 phenol in 5 minutes, nor by 1-120 in 15 minutes. 



A "B. diphtheriae phenol coefhcient" can then be calculated in the usual way from 

 the data obtained with the disinfectant and phenol if desired. 



B. TUBERCULOSIS 



Specimens of sputum from clinically active tuberculosis are pooled and evenly 

 mixed by prolonged shaking with broken glass. Stained smears of this material should 

 show one or more tubercle bacilli in nearly every field of the microscope or an average 

 of one to the field. 



Four 5-cc. portions of the pooled sputum are then measured out into sterile rubber- 

 stoppered bottles, and equal quantities of disinfectant in saline dilutions of 1-25, 1-50, 

 I-IOO, and 1-200 added, so that the final dilutions are as follows: 



'Reddish, George F.: op. cit., 17, 320. 1927. 



